Facile Fabrication of Surface Imprinted Macroporous Film for

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Functional Nanostructured Materials (including low-D carbon)

Facile Fabrication of Surface Imprinted Macroporous Film for Chemosensing of Human Chorionic Gonadotropin Hormone Marcin Dabrowski, Agnieszka Ziminska, Jakub Kalecki, Maciej Cieplak, Wojciech Lisowski, Radoslaw Maksym, Shuai Shao, Francis D'Souza, Alexander Kuhn, and Piyush Sindhu Sharma ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b17951 • Publication Date (Web): 04 Feb 2019 Downloaded from http://pubs.acs.org on February 6, 2019

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ACS Applied Materials & Interfaces

Facile Fabrication of Surface Imprinted Macroporous Film for Chemosensing of Human Chorionic Gonadotropin Hormone Marcin Dąbrowski,a Agnieszka Ziminska,a,b Jakub Kalecki,a Maciej Cieplak,a,* Wojciech Lisowski,a Radoslaw Maksym,c Shuai Shao,d Francis D’Souza,d Alexander Kuhn,e Piyush S. Sharmaa,* a

Institute of Physical Chemistry, Polish Academy of Sciences, Kasprzaka 44/52, 01-224

Warsaw, Poland b

Department of Biomaterials Chemistry, Faculty of Pharmacy with Laboratory Medicine

Division, Medical University of Warsaw, Banacha 1, 02-091, Warsaw, Poland c

Center of Postgraduate Medical Education, Department of Reproductive Health,

St. Sophia Hospital, Zelazna 90, 01-004 Warsaw, Poland d

Department of Chemistry, University of North Texas, 1155 Union Circle No. 305070, Denton,

TX 76203-5017, United States e

Univ. Bordeaux, CNRS UMR 5255, Bordeaux INP, ENSCBP, 16 Avenue Pey Berland, 33607

Pessac, France

ACS Paragon Plus Environment

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ABSTRACT

We present an improved approach for the preparation of highly selective and homogeneous molecular cavities in molecularly imprinted polymers (MIP) via the combination of surface imprinting and semi-covalent imprinting. Towards that, first a colloidal crystal mold was prepared via the Langmuir-Blodgett technique. Then, human chorionic gonadotropin (hCG) template protein was immobilized on the colloidal crystal mold.

Later, hCG derivatization with

electroactive functional monomers via amide chemistry was performed. In a final step, optimized potentiostatic polymerization of 2,3′-bithiophene enabled depositing an MIP film as macroporous structure. This synergistic strategy resulted in the formation of molecular imprinted cavities exclusively on the internal surface of the macropores, which were accessible after dissolution of silica molds. The recognition of hCG by the macroporous MIP film was transduced with the help of electric transducers, namely extended-gate field-effect transistors (EG-FET) and capacitive impedimetry (CI). These readout strategies offered the ability to create chemosensors for the labelfree determination of hCG hormone. Other than the simple confirmation of pregnancy, hCG assay is a common tool for the diagnosis and follow-up of ectopic pregnancy or trophoblast tumors. Concentration measurements with these EG-FET and capacitive impedimetry based devices allowed real-time measurements of hCG in the range of 0.8 to 900 fM and 0.17 to 2.0 fM, respectively in 10 mM carbonate buffer (pH = 10). Moreover, the selectivity of the chemosensors with respect to protein interferences was very high.

KEYWORDS: Molecularly imprinted polymer, Macroporous electrodes, Semi-covalent imprinting, Extended gate field effect transistor, Capacitive impedimetry, Chemosensor, Human chorionic gonadotropin hormone


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1. INTRODUCTION Molecularly imprinted polymers (MIPs) are frequently used nowadays to generate synthetic receptors having high binding affinity.1-2

These polymeric receptors are prepared via the

polymerization of functional and cross-linking monomers in the presence of target analyte.1 Removal of the template from the resulting polymeric material yields MIPs with specific molecular cavities for the template molecules, which are complementary in size, shape, and binding sites. As this material is synthesized chemically and provides a reasonably high binding efficiency, similar to enzymes/antibodies, they are called synthetic antibodies.3-6

The high binding efficiency

stimulated the frequent use of MIPs in chemosensing.7-10 Moreover, integration of MIPs into different 2-D materials resulted in materials with a sophisticated surface structure.11-16 However, the introduction of selectivity and binding efficiency, similar to biomimetic materials, in synthetic receptors is an arduous task,1-2 especially when target analytes are bulky and sensitive to the physico-chemical environment, such as proteins.17-20

One approach which is quite

successful in imprinting of bulky proteins and widely adopted by the imprinting community is “surface imprinting”.18, 21-23 The universal approach of surface imprinting is divided into two steps; the first step involves immobilization of the protein template on a surface, followed by polymerization around the immobilized template. The latter step appears more important because the thickness of the polymer is directly related to the success of imprinting. A very careful control of the polymerization conditions is necessary to obtain a MIP thickness of ~10-20 nm, compatible with the diameter of the protein template molecules.24 The main drawback of this approach appears to be the shelf-life of such thin hydrogel like polymers. There is a constant risk to lose the binding efficiency of MIPs during use or with time.


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Another approach for generating homogenous binding sites in molecular cavities is semicovalent imprinting. This methodology is well established for the imprinting of small molecules,2526

however, its application towards protein imprinting is not much studied.27-29 Recently, we

proposed an innovative route for protein imprinting.28 Our strategy is based on the synergy of three methodologies, namely inverse opal structuring, surface imprinting, and semi-covalent imprinting. Silica beads were used as a mold to prepare organized macroporous inverse opal polymeric films. To benefit from the advantage of semi-covalent imprinting, the human serum albumin (HSA) template was derivatized with functional monomers via C-N coupling chemistry27 prior to the immobilization on the silica mold.28 Later, these functionalized HSA templates were electrochemically cross-linked with an excess of cross-linking monomer. Removal of the silica mold created precisely defined cavities at the internal surface of the porous film. The binding and recognition performance of this macroporous structure was high.28 The bottle neck of this approach appeared to be the necessity of introducing a chromatographic purification step to separate derivatized HSA protein from unreacted monomers and side products before immobilization. This procedure also requires a large amount of protein. In the current work, we introduced a significant change to the surface imprinting procedure. Instead of immobilizing the already derivatized protein template, unmodified protein templates were immobilized first on the assembled silica beads.

After confirmation of successful

immobilization, these template molecules were then derivatized in two separate steps with functional monomers. In the final step of imprinting, an excess of cross-linker was used to build the polymer framework. It allowed to keep precisely the orientation of the binding sites in the molecular cavities. After removal of the protein templates and silica beads, a highly porous structure was developed. Such a porous structure was advantageous for non-restrictive diffusion


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of bulky protein towards the respective molecular cavities. The suitability and versatility of the developed imprinting approach was confirmed by devising MIP based electric chemsosensing systems for the determination of the human chorionic gonadotropin (hCG) hormone. Samples at each stage of sensor fabrication were characterized by X-ray photoelectron spectroscopy (XPS) to prove protein immobilization, template derivatization with functional monomers, imprinting and template extraction. Microscopic imaging by SEM confirmed a 3D macroporous film structure. The recognition of hCG by the macroporous MIP film was transduced with the help of electric transducers, namely extended-gate field-effect transistors (EG-FET) and capacitive impedimetry (CI). The proximity of the receptor with respect to the electrode surface is considered as the most critical step in the development of chemosensors. Therefore, electrodeposition was employed to prepare the MIP based recognition unit close to the transducer surfaces. The structures obtained via silica molding were advantageous to provide a well-organized reproducible macroporous structure, resulting in femtomolar sensitivity towards hCG determination in aqueous and synthetic serum samples. The most important advantage of a regular packing of silica beads in a colloidal crystal is a better control of the polymer growth between the silica beads. The clear current oscillations are only observed when a regular packing is obtained. The absence of clear current oscillations can result in overgrowth of polymer, and as a consequence eventually a closed pore structure, because it is more difficult to follow the progress of the polymer growth front in the template. The hCG hormone comprises α and β-subunits (Scheme 1).

Its molecular weight and

dimensions are Mw = 39.5 kD and 7.5 x 3.5 x 3.0 nm3, respectively.30 It is expressed by placental trophoblasts during pregnancy.31 Blood concentrations of hCG in the range of 5-25 mIU/mL are indicative of early pregnancy. Levels above 25 mIU/mL confirm pregnancy. Additionally, the


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hormone is a marker of many advanced malignancies. Therefore, it can assist in cancer detection.32 hCG assay is also a common tool for the diagnosis and follow-up of ectopic pregnancy or trophoblastic tumors and is a component of double and triple tests used in prenatal medicine to screen for fetal congenital abnormalities.33 Determination of the hCG hormone protein is performed mostly by different kinds of immunoassays

including

radio,34

electrochemical approaches.38-42

enzyme-linked35

electrochemiluminescent36-37

and

Other than immunoassays, HPLC combined with mass

spectrometry (HPLC-MS) has been often used as a laboratory based approach for the determination of this pregnancy hormone.43 This method out performs traditional techniques used for hormone testing because results provided by this technique are very selective with high throughput capabilities.44 However, a growing market demands easy-to-operate diagnosis tools. The HPLC-MS based determinations need trained personnel and they can only be performed in clinical laboratories. Aptamers in combination with nanoparticles were also proposed as a labelfree method for the highly sensitive determination of hCG.45-47 Label-based assays require a high degree of development to assure that the label does not block an important active site of the tagged molecule or modifies the molecule’s confirmation. Particularly for sandwich-type assays, both washing conditions and protocols of exposure to several reagents must intensively be optimized. Moreover, the high diversity in hormone levels reduces the value of single test analysis. Thus hormone profiling is needed. Therefore, introduction of an easy-to-operate, user-friendly, and cost-effective test kit for point-of-care (POC) diagnosis can increase the effectiveness and reduce costs of fertility problem treatments. Up to now only one report described the development of electrochemical MIP sensor for the hCG determination,48 based on the imprinting of hCG protein in an electroactive polydopamine film.


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2. MATERIALS AND METHODS 2.1. Chemicals. Silica beads (500 nm) were purchased from Fiber Optic Centre Inc. Electrochemical grade propylene carbonate solvent, HSA, cytochrome c, myoglobin, 2,2’bithiophene-5-carboxylic acid, 2,3’-bithiophene and LiClO4 supporting electrolyte were purchased from Sigma-Aldrich. N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC), 1hydroxy-7-azabenzotriazole (HOAt) and triethylamine were purchased from TCI chemicals. Synthetic details on the preparation of the bis(2,2′-bithienyl)-(4-aminophenyl)methane functional monomer, used to derivatized hCG, is given elsewhere.49 Analytical grade NaOH, Na2CO3, NaHCO3 and KF were procured from CHEMPUR. Pregnyl (N. V. Organon, 5000 IU) commercial drug was used as a source for hCG protein hormone. Merional (IBSA Institut Biochimique SA, 75 IU) was used as a source of follicle stimulating hormone (FSH) and luteinizing hormone (LH) in equimolar amounts. All polymer samples, as well as colloidal crystals were deposited on glass slides coated with thin layers of Au (150 nm) evaporated on Ti (15 nm) underlayers (Au-glass slides). 2.2. Instruments. A Zepto model plasma cleaner (100 W) from Diener Electronic was used for cleaning and hydrophilizing the surfaces of the Au plates. A rectangular (7 × 75) cm2 Langmuir trough type BAM 601 with accessories from Nima Technology, controlled by NIMA TR620 software, was used to perform the LB transfer of silica beads. For centrifugation of silica beads, a MPW Medical Instruments centrifuge model MPW-351R with the rotor No. 11457 was used. MIP films were imaged with scanning electron microscopy (SEM) using a Nova NanoSEM 450 microscope of the FEI Nova. Electropolymerization under potentiostatic conditions and capacitance measurements were performed using a SP-300 BioLogic potentiostat controlled by EC-Lab BioLogic software. A conical shaped glass cell was used for electrochemical experiments.


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A Au-glass slide, a Ag|AgCl pseudo reference electrode, and a Pt plate were used as the working, reference, and auxiliary electrode, respectively. A PHI 5000 VersaProbe™ (ULVCPHI) scanning ESCA microprobe using monochromatic A1 Kα radiation (hν = 1486.4 eV) was used to record XPS spectra. The XPS data were generated by a 100-μm diameter X-ray beam and collected from a 250-μm2 irradiated area. High-resolution XPS spectra were collected with a hemispherical analyzer at the pass energy of 23.5 eV, energy step of 0.1 eV, and photoelectron take off angle of 45° with respect to the surface plane. XPS data were analyzed with CASA XPS software. The binding energy of the Au 4f7/2 peak (BE = 84.0 eV) was chosen as an internal reference. The Shirley method was used to subtract background and peaks were fitted using the mixed Gaussian Lorentzian method. A Keithley 2636A (Keithley Instruments, Inc. OH, USA) dual-channel source meter was used for recording the characteristics. 2.3. Au surface preparation. Before assembling of the silica beads, Au-glass slides were hydrophilized by oxygen plasma treatment (50 W, 0.25 kPa, 10 min).50 Such a treatment in the presence of oxygen gas eliminates organic contaminants through chemically reacting with highly reactive radicals. Figure S1 shows the change of the Au contact angle from 73° to 32°. This change confirmed the increase in hydrophilicity of the Au surface after plasma treatment. 2.4. Fabrication of artificial opals deposited on the surface of Au-glass slides via the Langmuir-Blodgett technique.

Colloidal crystals were formed according to a previously

described procedure using the Langmuir-Blodgett (LB) technique.51-54 For that purpose, silica beads (500 nm), surface modified with 3-aminopropyltriethoxysilane, were washed at least five times with ethanol. Subsequently, a bead suspension was prepared in a mixed solvent solution of anhydrous ethanol and chloroform (1 : 4, v : v) at a bead concentration of ~10 mg/mL. The film


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was prepared on the air-water interface of the Langmuir trough filled with Milli-Q water. After evaporation of the solvents, the beads floating at the air-water interface were compressed with a barrier speed of 10 cm2/min to the target LB transfer pressure of 7 mN/m. Subsequently, this film was transferred onto an Au coated glass slide using the LB technique during emersion with a dipper speed of 1 mm/min. After the first withdrawal, the deposited crystal was dried and heated to promote adhesion of the silica beads of the innermost layer to the Au electrode surface.28 By repeating this procedure, an organized multilayer was produced on the Au coated glass slides (Scheme 2 Step I). 2.5. Immobilization of hCG protein hormone on the surface of the colloidal crystals. In order to immobilize hCG on the surface of a colloidal crystal, plates modified with a silica bead crystal were immersed in a 2.5% glutaraldehyde solution in phosphate buffer saline (pH = 7.4) for 1 h at room temperature. Then, the plates were washed three times with distilled water. Finally, the plates were immersed overnight at 4° C in a solution of hCG (3 mg/mL) in phosphate buffer saline (pH = 7.4) (Scheme 2 Step II). A series of washing with phosphate buffer saline (pH = 7.4) and Milli-Q water was performed three times to remove not immobilized protein molecules. 2.6. Derivatization of hCG protein with functional monomers.

The hCG molecules

immobilized on the surface of the colloidal crystal were then modified with two functional monomers, namely 2,2'-bithiophene-5-carboxylic acid and p-bis(2,2'-bithieny-5-yl)methylaniline (Scheme 2, Step III). For this purpose, plates with the hCG modified SiO2 colloidal crystal were immersed for 1 h into a solution of 2,2'-bithiophene-5-carboxylic acid (11 mg, 52 µM), EDC (15 μL), HOAt (11 mg, 80 µM) and triethylamine (25 μL) in a mixture of anhydrous toluene and anhydrous methylene chloride (1 mL, 7 : 3 v : v). These plates were then washed three times with anhydrous toluene to remove unreacted functional monomers and reactants. Subsequently, plates


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were immersed for another hour into a solution of p-bis(2,2'-bithieny-5-yl)methylaniline (20 mg, 46 µM), EDC (15 μL), triethylamine (25 μL) and HOAt (11 mg, 80 µM) in a mixture of anhydrous toluene and anhydrous methylene chloride (1 mL, 7 : 3 v : v). At the end, the plates were first washed with toluene three times, then three times with Mili-Q water (18 MΩ cm at 25 °C) and finally with phosphate buffer saline (pH = 7.4). 2.7. Electrochemical deposition of MIP on Au-glass slides modified with colloidal crystals with immobilized hCG template derivatized with functional monomers.

Optimized

potentiostatic conditions (1.25 V vs. Ag|Ag pseudo-reference electrode) were used to prepare a MIP film on the surface of an Au-glass slide (Scheme 2, Step IV). A propylene carbonate solution of 0.5 M 2,3’-bithiophene (the cross-linking monomer), and 3.5 M LiClO4 (the supporting electrolyte) was used for the electrosynthesis of MIP and NIP films. The Au-glass slides, coated with the monomer derivatized hCG modified colloidal crystal, served as a working electrode during this deposition. The polymerization was continued until +70 mC of charge had passed. After electropolymerization, the MIP films were rinsed with methanol to remove non reacted cross-linking monomer and the electrolyte, and then dried in air. As a control, a non-imprinted polymer (NIP) film was deposited on the template-free colloidal crystal using a similar solution composition and electropolymerization conditions. For the removal of the silica beads, slides were dipped for 5 min in 5% HF. After removal of the colloidal crystal, template protein was removed by washing with 30% NaOH solution (Scheme 2, Step V). 2.8. Determination of the electrical chemosensors analytical performance. The hCGextracted macroporous MIP coated Au-glass slides were used for hCG determination by CI under batch conditions. In all measurements, a macroporous MIP modified Au-glass slide (Scheme S1), a Pt plate and an Ag|AgCl wire served as the working, auxiliary and reference electrodes,


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respectively. The applied frequency and potential was hold at f = 10 Hz and Eappl = 0.20 V vs the Ag|AgCl pseudo-reference electrode, respectively. No faradaic process occurred at this potential. The hCG samples were prepared with a solution of the same composition as the binding solution, i.e., 10 mM carbonate buffer (pH = 10). The EG-FET sensing setup consisted of two main units (Scheme S1a). The recognition unit was deposited electrochemically on an Au-glass slide in the form of a macroporous MIP film (Scheme S1b and c). This extended recognition unit was electrically connected to the transduction unit, i.e., the gate of a commercial MOSFET model CD4007UB. The Pt plate played the role of a reference electrode. Details of the sensing system are given elsewhere.55-56 A 10 mM carbonate buffer solution (pH = 10) was used as a binding solution under batch conditions. Macroporous MIP films were washed with 30% NaOH solution after each binding experiment, and stored in 10 mM carbonate buffer solution (pH = 10) at room temperature.

3. RESULTS AND DISCUSSION 3.1. Fabrication of colloidal crystals on Au-glass slides via LB technique. LB deposition is a well-known tool to coat surfaces with ordered molecular films with a densely packed structure and precisely controlled thickness. This process involves the controlled compression of molecular monolayers formed by spreading of amphiphilic compounds at the air water interface, so as to induce ordering. In this process, a substrate, which is usually treated to have a hydrophilic surface, is initially immersed in water, and is drawn slowly upwards to transfer a layer of amphiphilic molecules. By moving the substrate up and down, an organized multilayer is produced. This approach was used to prepare reproducibly well-organized films of silica nanomolds on Au-glass slides (Scheme 2, Step I). Towards that, monolayers of silica nanobeads on the water surface were


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compressed. The resulting compression isotherm is shown in Fig. 1. An irreversible destruction of the film was observed above surface pressures of ~12 mN m-1. No phase transition in the monolayer was observed below this surface pressure. Thus, a surface pressure of 7 mN m-1 was selected to transfer a compressed organized layer of silica beads as a two-dimensional solid. This transfer was repeated four times to coat the Au-glass slides with a four-layer film of closely packed silica beads (Scheme 2, Step I). 3.2. Surface immobilization and derivatization of hCG protein with functional monomers. In the current approach of surface imprinting, efforts were made to improve the positioning of the binding sites in the molecular cavities of the MIP film. A combination of semi-covalent imprinting and surface imprinting is suitable for the generation of reproducible homogeneous molecular cavities with a precise orientation of the binding sites. For this, hCG protein was immobilized on the assembled n-aminopropyl functionalized silica beads (Scheme 2 Step I) via glutaraldehyde (Scheme 2 Step II). This immobilization technique using glutaraldehyde as the coupling agent is quite simple, efficient and probably the most used technique to carry out protein immobilization.5759

Moreover, by controlling the conditions, an activation of all the amino groups of the silica

support was possible. Glutaraldehyde reacted with different portions of the hCG, mainly involving the primary amino groups. For a precise control of the positioning of the binding sites in imprinted cavities of the MIP, surface immobilized hCG was derivatized with amine and carboxy functionalized bithiophene functional monomers via C-N coupling chemistry (Scheme 2, Step III). For the derivatization of hCG protein, the carbodiimide conjugation approach was used. It works by activating carboxyl groups for direct reaction with primary amines via amide bond formation. 3.3. MIP and NIP electrodeposition within the voids of a colloidal crystal. In the final imprinting step, the hCG molecules derivatized with functional monomers were cross-linked with


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poly(2,3′-bithiophene) (Scheme 2, Step IV).

For that purpose, 2,3′- bithiophene was

electropolymerized under potentiostatic conditions. The choice of poly(2,3′-bithiophene) was based on the fact that this monomer allows cross-linking more efficiently at lower oxidation potentials and gives more cross-linked films than other similar bithiophenes.28 This good crosslinking efficiency helped to create rigid and stable molecular cavities in the MIP (Scheme 2, Step V). The MIP electrodeposition conditions were carefully chosen to prepare uniform, compact and stable macroporous polymeric structures. It is known that the supporting electrolyte has minor effects, while the solvent has a very clear and significant influence on the morphology of the polymer. Continues films prepared in propylene carbonate have a smooth and flat morphology. However, films demonstrated a rough structure if prepared in acetonitrile.60 This makes this conditions not suitable for the deposition of macroporous films. Therefore, propylene carbonate was chosen as a solvent for electropolymerization. Similarly, it was found that films prepared under potentiodynamic conditions showed rougher structures than those prepared using a constant potential. Therefore, potentiostatic conditions were employed for film preparation. Furthermore, the composition of the electropolymerization solution (2,3’-bithiophene and supporting electrolyte concentrations) was optimized to obtain a stable, uniform macroporous structure. The detailed optimization is described in the Supporting Information (Fig. S2). Under optimized potentiostatic conditions, electrooxidation at 1.25 V resulted in the generation of a bithiophene cation radical, which then initiated the polymerization. Therefore, the crosslinking monomer was added at a relatively high concentration. Noticeably, functional monomers located on the hCG proteins co-polymerized with the poly(2,3′-bithiophene) matrix during this process, thus constituting a part of the polymer. With time, the poly(2,3′-bithiophene) started to


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grow from the electrode surface (bottom of the colloidal crystal) to the top of the colloidal crystal, thus filling the voids inside the colloidal crystals. Current oscillations during such a deposition helped to tune the thickness of the polymer film very precisely (Fig. 2 A).61 A clear oscillation in current during NIP electropolymerization indicates close packing of silica beads (Fig. 2 A2). It also proves that the electrodeposition is occurring from the bottom (electrode surface) to the top. Comparatively, current oscillations during MIP electropolymerization was less pronounced but still visible (Fig. 2 A1). This may originate from a rearrangement of the beads during functional monomer derivatization.

Additionally, the presence of the bithiophene moieties on the

hCG/(functional monomer) modified silica bead surface may also influence the deposition process. However, the final macroporous film obtained after removal of the silica beads, which serve as sacrificial molds for electropolymerization, exhibits a fairly organized and crack free morphology for both MIP and NIP films (Fig. 2B and Fig. S3). 3.4. SEM imaging of the colloidal crystals, macroporous MIP and NIP films. SEM imaging enables to investigate the structure of the colloidal crystals as well as the structure of the resulting porous MIP and NIP films (Fig. 2B and Fig. S3). Deposited colloidal crystals were composed of four layers of silica nanobeads (Fig. 2B 1, 2). SEM imaging also showed that the ordering of the beads in the first monolayer was the highest. However, the ordering in each following layer was deteriorated. Despite the defects in the structure, the ordering of the beads was sufficient for the subsequent fabrication of macroporous MIP and NIP films. Final macroporous MIP (Fig. 2B 3-4) and NIP (Fig. 2B 5-6) films showed porous structure. Both films had a thickness equal to 2.5 monolayers of silica beads. Importantly, it was clearly visible that the films had interconnected pores. Additionally, in both films the outer layers were fully open although the organization of the film was not perfect. This is crucial for the performance of the sensor. The generation of a 2.5


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monolayer porous structure correlates well with the current oscillations recorded during the potentiostatic polymerization of 2,3’-bithiophene (Fig. 2A). 3.5. XPS characterization of surface imprinting steps. Samples prepared in the different steps of surface imprinting were characterized by XPS (Fig. 3). Table S1 shows the atomic percentage of the most interesting elements present in different samples including LB assembled amine terminated silica beads (SiO2NH2) (Fig. 3A I), hCG immobilized with the help of glutaraldehyde over silica beads (SiO2NH2-Glu-hCG) (Fig. 3A II) and functional monomer derivatized hCG protein on silica beads (SiO2NH2-Glu-hCG-FM) (Fig. 3A III and Fig. 3B III), functional monomer derivatized hCG protein on silica beads cross-linked with MIP film (Fig. 3A IV and Fig. 3B IV), and the final macroporous MIP film (Fig. 3A V and Fig. 3B V). XPS spectra clearly confirm the successful immobilization of hCG protein on silica beads through glutaraldehyde chemistry. In comparison to SiO2NH2 (Fig. 3A I), a pronounced increase of the N 1s signal (Fig. 3A II) and the N atomic percentage (Table S1) was observed in SiO2NH2Glu-hCG sample. This N 1s signal increased further (Table S1) when the hCG was derivatized with amine and carboxy functionalized monomers (SiO2NH2-Glu-hCG-FM).

XPS spectra

indicated a pronounced drop of the N 1s signal and its atomic percentage when the hCG immobilized silica beads were coated with the poly(2,3′-thiophene) film (Fig. 3A IV. and Table S1). Finally, in the macroporous MIP sample, the N 1s signal increased again (Fig. 3A V). Such an increase originates from the amine moieties of the functional monomers which are located directly on the surface of the molecular cavities. Moreover, XPS spectra confirm the successful derivatization of hCG with functional monomers (Fig. 3B III) as evidenced by the appearance of characteristic S peaks between 164 and 168 eV. Such S peaks were also present in macroporous


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MIP (Fig. 3B V), confirming the successful electropolymerization of cross-linking monomer on the silica beads. 3.6. hCG determination by capacitive impedimetry. CI experiments were performed at a constant potential of 0.20 V and a potential amplitude of alternating current of 10 mV. During measurements, the frequency was kept constant at 10 Hz. It is known that, at such a low frequency, the MIP film coated electrode mainly reveals capacitive properties.62 This capacity was low at low potential and it rapidly increased with increasing potential. Therefore, a potential well below the oxidation potential of thiophenes, i.e. 0.20 V, was selected as an optimized potential in these measurements. Under batch conditions, with relatively high solution volume, it is important to improve the transport rate of analyte. Typically, convection is introduced into the system to increase such transport. Therefore, during the measurements, the solution was stirred at a constant speed to provide convection. Combining controlled convection with diffusion increases the transport rate of the analyte to the electrode surface, thus allowing determination of analytes at lower concentrations. A sufficiently low concentration of carbonate buffer solution was equilibrated with the working electrodes before injection of hCG. A stable electrical double layer was evidenced by constant capacitance values obtained as a function of time. When hCG was introduced in the binding solution, the proteins bind to the molecular cavities present at the internal surface of the macroporous film. The binding of hCG protein in the molecular cavities governs mostly through complementary electrostatic interactions and hydrogen bonding. Probably hydrophobicity of the imprinted cavity also plays role. This binding causes the increase in capacitance (Fig. 4A). The protein binding strongly affects the electric permittivity of this layer. In order to increase the


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sensitivity of this capacitance transduction, we used a relatively low concentration of supporting electrolyte (10 mM). At such low concentrations of supporting electrolyte, it was possible to obtain a sufficiently thick double layer. A thin double layer can compromise the chemosensor sensitivity by leaving a major portion of the analyte outside. The determined capacitance increases with further addition of different portions of hCG (Fig. 4A). The linear dynamic concentration range extended from 0.17 to 2.0 fM hCG with the calibration plot following a linear regression equation of capacitance ΔC [nF] = 63.3 (±3.1) [nF/fM]  chCG [fM] + 7.88 (±3.2) [nF].

The

correlation coefficient was R2 = 0.976 while the LOD and sensitivity at the signal-to-noise ratio (S/N) of 3 was 0.17 fM and 63.3 (±3.1) [nF/fM], respectively. An additional experiment was performed to confirm the formation of molecular cavities in macroporous MIP. For that purpose, a non-imprinted macroporous polymer (NIP) film deposited as a control on a gold electrode was used under the same conditions as those for MIP. Interestingly, when a macroporous NIP film was used under similar conditions to determine hCG, the results were very much different than those obtained for MIP. There was no change in capacitance after addition of hCG protein (Fig. 4B). This confirms the high efficiency of the imprinting process. A cross-selectivity study was performed in the presence of a mixture of LH and FSH protein (Fig. 4C). The result showed a linear change in capacitance of the MIP film when different portions of LH and FSH protein samples were allowed to bind. These protein hormones have very similar mass, size, shape and structure as hCG.

However, these changes were inferior in

comparison to changes observed in the case of hCG binding. The sensitivity of the macroporous MIP film towards LH-FSH protein was 30.3±3.2 nF/fM (Fig. 4C). Additional cross-reactivity experiments confirmed the selectivity of the devised chemosensor. Towards that, the MIP film capacitance was measured in the presence of common proteins present


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in blood serum samples, such as cytochrome c, myoglobin and HSA (Fig. S4-S6). During binding studies, for better comparison, the concentration of these proteins was kept in the same order as for hCG. These capacitance changes during interfering protein binding were less pronounced in comparison to changes obtained during hCG binding. The sensitivity of the macroporous MIP film towards cytochrome c, myoglobin and HSA proteins was 1.7±1.1 nF/fM, 3.1±1.3 nF/fM, and 26.5±2.7 nF/fM, respectively (Fig. 4D). In a final step of chemosensor development, the system was used for determination of different concentrations of hCG in a synthetic serum sample (Fig. S7). For that, a commercial NORTROL serum was diluted 1000 times with the 10 mM carbonate buffer solution (pH = 10). Interestingly, the chemosensor was able to determine hCG in diluted synthetic serum samples (Fig. S7). Moreover, the slope of the constructed calibration curve (50.1±1.4 nF/fM) was very similar to the slope of the calibration curve obtained in aqueous solution (63.3±3.1 nF/fM). This result confirms the suitability of the devised chemosensor for hCG determination in real samples. 3.7. hCG determination using extended gate field effect transistor. An EG-FET is a modified form of a classical chemFET, in which a chemically-sensitive recognition unit is deposited on the end of the signal line extended from the classical field effect transistor gate. Therefore, any changes of potential at the extended surface transferred to the transistor gate results in changes of source-drain current. This change is exactly the same as if the potential change would take place at the gate itself. Importantly, there is a need to apply a certain voltage across the solution|polymer|Au-glass interfaces in order to drive the transistor gate open or closed. The details of the working hypothesis of this novel system is described in many recent reports.28, 56, 63 The binding efficiency of hCG protein to the macroporous MIP films was studied by the EGFET in different solutions of low salt concentrations. The measurement conditions were optimized


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to enhance interactions of analyte and its molecular cavities. Different binding solutions, such as distilled water, 10 mM phosphate buffer (pH = 7.0) and 10 mM carbonate buffer (pH=10) were examined. Except for carbonate buffer, in other binding solutions the changes in source-drain currents after addition of hCG protein were indistinguishable from noise.

The EG-FET

transduction method is sensitive to charged species. Therefore, protein analytes can be determined only at pH values that are far from their isoelectric points. The protein concentration dependent binding experiment in 10 mM carbonate buffer solution demonstrated a dynamic concentration range from 0.8 to 900 fM hCG. A plot of the drain current change vs log hCG concentration (Fig. 5A) in this concentration range was obeying the linear regression equation ΔI [µA] = 1.78  10-6

(±0.12  10-6) [µA/log M] log(chCG, M) + 2.6  10-5

(±0.2  10-5) [µA].

The

correlation coefficient was R2 = 0.950 while the LOD and sensitivity at the signal-to-noise ratio (S/N) of 3 was 0.8 fM and –1.8  10-6 (±0.12  10-6) µA (log M)-1, respectively. The effect of imprinting and the presence of molecular cavities was clearly evident when the binding performance of a macroporous NIP film was studied in the similar concentration range as a control. The change of drain current after hCG binding was much smaller than the change obtained at a macroporous MIP film (Fig. 5B). A quite different behavior of the NIP coated electrodes in both transduction methods, namely EG-FET (Fig. 5B) and CI (Fig. 4B) was observed. Presumably, the nonspecific adsorption of hCG on the NIP top surface may not influence the electrical permittivity of the polymer film. However, it may strongly affect the potential of the FET gate. The chemosensor selectivity with respect to common interfering proteins, such as cytochrome c, myoglobin, HSA and low-mass bio-compounds including creatinine, creatine, glucose and urea was quite high (Fig. 5A and C). The sensitivity of the macroporous MIP film towards cytochrome


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c, myoglobin and HSA proteins was -0.40  10-6 (±0.06  10-6) µA (log M)-1, -0.39  106

(±0.13  10-6) µA (log M)-1, and -0.85  10-6 (±0.24  10-6) µA (log M)-1, respectively (Fig. 5A).

The good selectivity is originating from the designed geometric and dynamic features of the MIP that is complementary exclusively for the desired analyte, but not for other possible interfering substrates. Even though hCG protein contains significantly less sites for functional monomer derivatization than the HSA protein studied before,27-28 the higher selectivity of MIP confirmed the versatility of our approach of surface imprinting. The devised chemosensor was further tested in control Nortrol serum sample (Fig. 5D). Spiking the control serum sample with hCG allowed the measurement of known concentrations of hCG in the 1000 times diluted control Nortrol serum sample. The sensitivity of the chemosensor in control serum sample (-2.2  10-6 [±0.12  10-6] µA [log M]-1) was very similar to the sensitivity in an aqueous system (-1.8  10-6 [±0.12  10-6] µA [log M]-1). This result confirms the suitability of the chemosensor to determine hCG in real samples after dilution. Table 1 summarizes the analytical parameters of some representative bio- and chemosensors reported in the last decade.45-48, 64-68 These methods reported trace level determination of hCG protein in real samples. Compared to these electrochemical46, 48, 64 and optical sensors,45, 47, 67 the detectability obtained in the present work is in the fM range, which is well below concentrations expected to be found in samples originating from real patients. 3.8. Signal repeatability and stability test of chemosensors. The signal repeatability was verified by repeating each experiment at least three times. The data of such measurements were used to plot a graph with error bars (Fig. 4 and 5). In parallel, the performance of both chemosensors was tested as a function of time. It was observed that the chemosensor response towards hCG protein was stable (Fig. S8). The stability was checked by measuring the 1.6 fM


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(capacitive) and 19 fM hCG solutions (for EG-FET) (Fig. S8). The response signal obtained during a one-week period from both chemosensors was very similar. The standard deviation of the EGFET chemosensor for 19 fM was 0.48 µA with a variance of 0.23 µA. Similarly, the standard deviation of the capacitance chemosensor response for 1.6 fM was 15 nF with a variance of 0.24 nF.

4. Conclusions We presented an improved approach for surface imprinting of protein macromolecules. The combination of semi-covalent imprinting with surface imprinting and the use of inverse opal structures appeared to be efficient in the development of highly sensitive sensing system. Semicovalent imprinting helped to generate precise molecular cavities in the macroporous MIP film. This macroporous MIP was combined successfully as a recognition element with two different transduction techniques, namely capacitive and FET based sensing. These readout strategies offer the ability to develop chemosensors for the label-free determination of hCG hormones. Concentration dependent determinations made with EG-FET and capacitance based devices exhibit real-time reversible signals for hCG from 0.8 to 900 fM and 0.17 to 2.0 fM, respectively. This is well below the concentrations which are expected to be found in samples originating form real patients. Thus, the presented approach opens up very interesting perspectives for the fast and reliable screening of real clinical samples. ASSOCIATED CONTENT Supporting Information. Contact angle measurements, Optimization of polymerization conditions, Table summarizing XPS analysis results of different samples prepared during surface imprinting, SEM images of macroporous MIP and NIP film (at low magnification), Interferents as


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well as hCG determination by capacitive impedimetry, Stability test, Scheme of EG-FET experimental setup and optical images of gold plates modified with silica colloidal crystals and macroporous polymer are described in Supporting Information. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] (P.S.S.). *E-mail: [email protected] (M.C.). Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ACKNOWLEDGEMENTS The present research was financially supported by the National Science Centre (Grant No. NCN 2017/25/B/ST4/01696 to P.S.S.). Authors are thankful to Dr. Adam Leśniewski (IPC PAS, Warsaw, Poland) for contact angle measurements. Authors are also thankful to Dr. Marcin Holdynski (IPC PAS, Warsaw, Poland) for SEM measurements. F.D. acknowledges financial support by the UNT-AMMPI.

ABBREVIATIONS CI EDC EG-FET fM FM FSH

capacitive impedimetry N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride extended-gate field effect transistor femtomolar functional monomers follicle stimulating hormone


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hCG HOAt HPLC-MS HSA LB LH MIP MOSFET nF NIP POC SEM SiO2-NH2 SiO2-NH2-Glu-hCG SiO2-NH2-Glu-hCG-FM SiO2-NH2-Glu-hCG-MIP XPS

human chorionic gonadotropin hormone 1-hydroxy-7-azabenzotriazole high performance liquid chromatography-mass spectrometry human serum albumin Langmuir-Blodgett luteinizing hormone molecular imprinted polymer metal-oxide-semiconductor field effect transistor nano Farad non-imprinted polymer point-of-care scanning electron microscopy LB assembled amine terminated silica beads hCG immobilized with the help of glutaraldehyde over silica beads functional monomers derivatized hCG protein immobilized on silica beads 2,3’-bithiophene co-polymerized with functional monomers in between hCG protein modified silica beads X-ray photoelectron spectroscopy

REFERENCES (1) Cieplak, M.; Kutner, W. Artificial Biosensors: How Can Molecular Imprinting Mimic Biorecognition? Trends Biotechnol. 2016, 34, 922-941. (2) Sharma, P. S.; Iskierko, Z.; Pietrzyk-Le, A.; D'Souza, F.; Kutner, W. Bioinspired Intelligent Molecularly Imprinted Polymers for Chemosensing: A Mini Review. Electrochem. Commun. 2015, 50, 81-87. (3) Sharma, P. S.; Iskierko, Z.; Noworyta, K.; Cieplak, M.; Borowicz, P.; Lisowski, W.; D'Souza, F.; Kutner, W. Synthesis and Application of a “Plastic Antibody” in Electrochemical Microfluidic Platform for Oxytocin Determination. Biosens. Bioelectron. 2018, 100, 251-258. (4) Takeuchi, T.; Kitayama, Y.; Sasao, R.; Yamada, T.; Toh, K.; Matsumoto, Y.; Kataoka, K. Molecularly Imprinted Nanogels Acquire Stealth In Situ by Cloaking Themselves with Native Dysopsonic Proteins. Angew. Chem. 2017, 129, 7194-7198. (5) Hoshino, Y.; Kodama, T.; Okahata, Y.; Shea, K. J. Peptide Imprinted Polymer Nanoparticles: A Plastic Antibody. J. Am. Chem. Soc. 2008, 130, 15242-15243. (6) Luan, J.; Liu, K.-K.; Tadepalli, S.; Jiang, Q.; Morrissey, J. J.; Kharasch, E. D.; Singamaneni, S. PEGylated Artificial Antibodies: Plasmonic Biosensors with Improved Selectivity. ACS Appl. Mater. Interfaces 2016, 8, 23509-23516. (7) Uzun, L.; Turner, A. P. F. Molecularly-Imprinted Polymer Sensors: Realising Their Potential. Biosens. Bioelectron. 2016, 76, 131-144. (8) Yáñez-Sedeño, P.; Campuzano, S.; Pingarrón, J. M. Electrochemical Sensors Based on Magnetic Molecularly Imprinted Polymers: A Review. Anal. Chim. Acta 2017, 960, 1-17.


23


Page 24 of 38

(9) Zamora-Gálvez, A.; Morales-Narváez, E.; Mayorga-Martinez, C. C.; Merkoçi, A. Nanomaterials Connected to Antibodies and Molecularly Imprinted Polymers as Bio/Receptors for Bio/Sensor Applications. Appl. Mater. Today 2017, 9, 387-401. (10) Peltomaa, R.; Benito-Peña, E.; Moreno-Bondi, M. C. Bioinspired Recognition Elements for Mycotoxin Sensors. Anal. Bioanal. Chem. 2018, 410, 747-771. (11) Atar, N.; Yola, M. L. Core-Shell Nanoparticles/Two-Dimensional (2D) Hexagonal Boron Nitride Nanosheets with Molecularly Imprinted Polymer for Electrochemical Sensing of Cypermethrin. J. Electrochem. Soc. 2018, 165, H255-H262. (12) Atar, N.; Yola, M. L.; Eren, T. Sensitive Determination of Citrinin Based on Molecular Imprinted Electrochemical Sensor. Appl. Surf. Sci. 2016, 362, 315-322. (13) Yola, M. L.; Atar, N. Electrochemical Detection of Atrazine by Platinum Nanoparticles/Carbon Nitride Nanotubes with Molecularly Imprinted Polymer. Ind. Eng. Chem. Res. 2017, 56, 7631-7639. (14) Yola, M. L.; Atar, N. A Novel Detection Approach for Serotonin by Graphene Quantum Dots/Two-Dimensional (2D) Hexagonal Boron Nitride Nanosheets with Molecularly Imprinted Polymer. Appl. Surf. Sci. 2018, 458, 648-655. (15) Yola, M. L.; Atar, N. A Highly Efficient Nanomaterial with Molecular Imprinting Polymer: Carbon Nitride Nanotubes Decorated with Graphene Quantum Dots for Sensitive Electrochemical Determination of Chlorpyrifos. J. Electrochem. Soc. 2017, 164, B223-B229. (16) Yola, M. L.; Göde, C.; Atar, N. Molecular Imprinting Polymer with Polyoxometalate/Carbon Nitride Nanotubes for Electrochemical Recognition of Bilirubin. Electrochim. Acta 2017, 246, 135-140. (17) Iskierko, Z.; Sharma, P. S.; Bartold, K.; Pietrzyk-Le, A.; Noworyta, K.; Kutner, W. Molecularly Imprinted Polymers for Separating and Sensing of Macromolecular Compounds and Microorganisms. Biotechnol. Adv. 2016, 34, 30-46. (18) Li, S.; Cao, S.; Whitcombe, M. J.; Piletsky, S. A. Size Matters: Challenges in Imprinting Macromolecules. Prog. Polym. Sci. 2014, 39, 145-163. (19) Muhammad, P.; Tu, X.; Liu, J.; Wang, Y.; Liu, Z. Molecularly Imprinted Plasmonic Substrates for Specific and Ultrasensitive Immunoassay of Trace Glycoproteins in Biological Samples. ACS Appl. Mater. Interfaces 2017, 9, 12082-12091. (20) Bertolla, M.; Cenci, L.; Anesi, A.; Ambrosi, E.; Tagliaro, F.; Vanzetti, L.; Guella, G.; Bossi, A. M. Solvent-Responsive Molecularly Imprinted Nanogels for Targeted Protein Analysis in MALDI-TOF Mass Spectrometry. ACS Appl. Mater. Interfaces 2017, 9, 6908-6915. (21) Zhang, X.; Yarman, A.; Erdossy, J.; Katz, S.; Zebger, I.; Jetzschmann, K. J.; Altintas, Z.; Wollenberger, U.; Gyurcsányi, R. E.; Scheller, F. W. Electrosynthesized MIPs for Transferrin: Plastibodies or Nano-Filters? Biosens. Bioelectron. 2018, 105, 29-35. (22) Jetzschmann, K. J.; Yarman, A.; Rustam, L.; Kielb, P.; Urlacher, V. B.; Fischer, A.; Weidinger, I. M.; Wollenberger, U.; Scheller, F. W. Molecular LEGO by Domain-Imprinting of Cytochrome P450 BM3. Colloids Surf. B 2018, 164, 240-246.


24

Page 25 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(23) Ouyang, J.; Liu, Z.; Han, Y.; Zeng, K.; Sheng, J.; Deng, L.; Liu, Y.-N. Fabrication of Surface Protein-Imprinted Biofuel Cell for Sensitive Self-Powered Glycoprotein Detection. ACS Appl. Mater. Interfaces 2016, 8, 35004-35011. (24) Dabrowski, M.; Lach, P.; Cieplak, M.; Kutner, W. Nanostructured Molecularly Imprinted Polymers for Protein Chemosensing. Biosens. Bioelectron. 2018, 102, 17-26. (25) Chen, L.; Xu, S.; Li, J. Recent Advances in Molecular Imprinting Technology: Current Status, Challenges and Highlighted Applications. Chem. Soc. Rev 2011, 40, 2922-2942. (26) Curcio, P.; Zandanel, C.; Wagner, A.; Mioskowski, C.; Baati, R. Semi-Covalent Surface Molecular Imprinting of Polymers by One-Stage Mini-emulsion Polymerization: Glucopyranoside as a Model Analyte. Macromol. Biosci. 2009, 9, 596-604. (27) Cieplak, M.; Szwabinska, K.; Sosnowska, M.; Chandra, B. K. C.; Borowicz, P.; Noworyta, K.; D'Souza, F.; Kutner, W. Selective Electrochemical Sensing of Human Serum Albumin by Semi-Covalent Molecular Imprinting. Biosens. Bioelectron. 2015, 74, 960-966. (28) Dabrowski, M.; Cieplak, M.; Sharma, P. S.; Borowicz, P.; Noworyta, K.; Lisowski, W.; D'Souza, F.; Kuhn, A.; Kutner, W. Hierarchical Templating in Deposition of Semi-Covalently Imprinted Inverse Opal Polythiophene Film for Femtomolar Determination of Human Serum Albumin. Biosens. Bioelectron. 2017, 94, 155-161. (29) Taguchi, H.; Sunayama, H.; Takano, E.; Kitayama, Y.; Takeuchi, T. Preparation of Molecularly Imprinted Polymers for the Recognition of Proteins via the Generation of PeptideFragment Binding Sites by Semi-Covalent Imprinting and Enzymatic Digestion. Analyst 2015, 140, 1448-1452. (30) Lapthorn, A. J.; Harris, D. C.; Littlejohn, A.; Lustbader, J. W.; Canfield, R. E.; Machin, K. J.; Morgan, F. J.; Isaacs, N. W. Crystal Structure of Human Chorionic Gonadotropin. Nature 1994, 369, 455-461. (31) Cole, L. A. Human Chorionic Gonadotropin and Associated Molecules. Expert Rev. Mol. Diagnostics 2009, 9, 51-73. (32) Cole, L. A. HCG Variants, the Growth Factors Which Drive Human Malignancies Am. J. Cancer Res. 2012, 2, 22-35. (33) Leostic, A.; Tran, P. L.; Fagot, H.; Boukerrou, M. Elevated Human Chorionic Gonadotrophin without Pregnancy: A Case of Gallbladder Carcinoma. J. Gynecology Obstet. Hum. Reprod. 2018, 47, 141-143. (34) Koninckx, P.; De Moor, P. A Rapid Radioimmunoassay of Human LH and HCG Useful in Early Pregnancy Problems. Eur. J. Obs. Gynecology Rep. Biol. 1976, 6, 239-241. (35) Kelly, L. S.; Kozak, M.; Walker, T.; Pierce, M.; Puett, D. Lectin Immunoassays Using Antibody Fragments to Detect Glycoforms of Human Chorionic Gonadotropin Secreted by Choriocarcinoma Cells. Anal. Biochem. 2005, 338, 253-262. (36) Liao, N.; Zhuo, Y.; Chai, Y.; Xiang, Y.; Cao, Y.; Yuan, R.; Han, J. Amplified Electrochemiluminescent Immunosensing Using Apoferritin-Templated Poly(ethylenimine) Nanoparticles as Co-Reactant. Chem. Commun. 2012, 48, 7610-7612.


25


Page 26 of 38

(37) Zhang, A.; Guo, W.; Keb, H.; Zhang, X.; Zhang, H.; Huang, C.; Yang, D.; Jia, N.; Cui, D. Sandwich-Format ECL Immunosensor Based on Au Star@BSA-Luminol Nanocomposites for Determination of Human Chorionic Gonadotropin. Biosens. Bioelectron. 2018, 101, 219-226. (38) Charoenkitamorn, K.; Tue, P. T.; Kawai, K.; Chailapakul, O.; Takamura, Y. Electrochemical Immunoassay Using Open Circuit Potential Detection Labeled by Platinum Nanoparticles. Sensors 2018, 18, 444. (39) Idegami, K.; Chikae, M.; Kerman, K.; Nagatani, N.; Yuhi, T.; Endo, T.; Tamiyae, E. Gold Nanoparticle-Based Redox Signal Enhancement for Sensitive Detection of Human Chorionic Gonadotropin Hormone. Electroanalysis 2008, 20, 14–21. (40) Li, P.; Ge, B.; Ou, L. M.-L.; Yao, Z.; Yu, H.-Z. DNA-Redox Cation Interaction Improves the Sensitivity of an Electrochemical Immunosensor for Protein Detection. Sensors 2015, 15, 2054320556. (41) Viet, N. X.; Chikae, M.; Ukita, Y.; Maehashi, K.; Matsumoto, K.; Tamiya, E.; Viet, P. H.; Takamura, Y. Gold-Linked Electrochemical Immunoassay on Single-Walled Carbon Nanotube for Highly Sensitive Detection of Human Chorionic Gonadotropin Hormone. Biosens. Bioelectron. 2013, 42, 592-597. (42) Yang, G.; Yang, X.; Yang, C.; Yang, Y. A Reagentless Amperometric Immunosensor for Human Chorionic Gonadotrophin Based on a Gold Nanotube Arrays Electrode. Colloids Surf. A: Physicochem. Eng. Asp. 2011, 389, 195-200. (43) Rosting, C.; Tran, E. V.; Gjelstad, A.; Halvorsen, T. G. Determination of the Low-Abundant Protein Biomarker hCG from Dried Matrix Spots Using Immunocapture and Nano Liquid Chromatography Mass Spectrometry. J. Chromatogr. B 2018, 1077-1078, 44-51. (44) Gronowski, A. M. Clinical Assays for Human Chorionic Gonadotropin. Clin. Chem. 2009, 55, 1900. (45) Chiu, N.-F.; Kuo, C.-T.; Lin, T.-L.; Chang, C.-C.; Chen, C.-Y. Ultra-High Sensitivity of the Non-immunological Affinity of Graphene Oxidepeptide-based Surface Plasmon Resonance Biosensors to Detect Human Chorionic Gonadotropin. Biosens. Bioelectron. 2017, 94, 351-357. (46) Xia, N.; Chen, Z.; Liu, Y.; Ren, H.; Liu, L. Peptide Aptamer-Based Biosensor for the Detection of Humanchorionic Gonadotropin by Converting Silver Nanoparticles-Based Colorimetric Assay into Sensitive Electrochemical Analysis. Sens. Actuators B 2017, 243, 784791. (47) Chang, C.-C.; Chen, C.-P.; Lee, C.-H.; Chen, C.-Y.; Lin, C.-W. Colorimetric Detection of Human Chorionic Gonadotropin Using Catalytic Gold Nanoparticles and a Peptide Aptamer. Chem. Commun. 2014, 50, 14443-14446. (48) Shen, X.; Ma, Y.; Zeng, Q.; Huang, J.; Tao, J.; Wang, L. Ultrasensitive Determination of Human Chorionic Gonadotropin Using a Molecularly Imprinted Electrochemical Sensor. Chemistry Select 2017, 2, 6549-6555. (49) Huynh, T. P.; Sosnowska, M.; Sobczak, J. W.; Kc, C. B.; Nesterov, V. N.; D'Souza, F.; Kutner, W. Simultaneous Chronoamperometry and Piezoelectric Microgravimetry Determination


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of Nitroaromatic Explosives Using Molecularly Imprinted Thiophene Polymers. Anal. Chem. 2013, 85, 8361-8368. (50) Karajić, A.; Reculusa, S.; Heim, M.; Garrigue, P.; Ravaine, S.; Mano, N.; Kuhn, A. Bottomup Generation of Miniaturized Coaxial Double Electrodes With Tunable Porosity. Adv. Mater. Interfaces 2015, 2, 1500192. (51) Ye, X.; Qi, L. Two-Dimensionally Patterned Nanostructures based on Monolayer Colloidal Crystals: Controllable Fabrication, Assembly, and Applications. Nano Today 2011, 6, 608-631. (52) Pernites, R. B.; Foster, E. L.; Felipe, M. J. L.; Robinson, M.; Advincula, R. C. Patterned Surfaces Combining Polymer Brushes and Conducting Polymer via Colloidal Template Electropolymerization. Adv. Mater. 2011, 23, 1287-1292. (53) Reculusa, S.; Ravaine, S. Synthesis of Colloidal Crystals of Controllable Thickness through the Langmuir−Blodgett Technique. Chem. Mater. 2003, 15, 598-605. (54) Szamocki, R.; Reculusa, S.; Ravaine, S.; Bartlett, P. N.; Kuhn, A.; Hempelmann, R. Tailored Mesostructuring and Biofunctionalization of Gold for Increased Electroactivity. Angew. Chem. Int. Ed. 2006, 45, 1317-1321. (55) Dabrowski, M.; Cieplak, M.; Noworyta, K.; Heim, M.; Adamkiewicz, W.; Kuhn, A.; Sharma, P. S.; Kutner, W. Surface Enhancement of a Molecularly Imprinted Polymer Film Using Sacrificial Silica Beads for Increasing L-Arabitol Chemosensor Sensitivity and Detectability. J. Mater. Chem. B 2017, 5, 6292-6299. (56) Iskierko, Z.; Checinska, A.; Sharma, P. S.; Golebiewska, K.; Noworyta, K.; Borowicz, P.; Fronc, K.; Bandi, V.; D'Souza, F.; Kutner, W. Molecularly Imprinted Polymer Based ExtendedGate Field-Effect Transistor Chemosensors for Phenylalanine Enantioselective Sensing. J. Mater. Chem. C 2017, 5, 969-977. (57) Zucca, P.; Sanjust, E. Inorganic Materials as Supports for Covalent Enzyme Immobilization: Methods and Mechanisms. Molecules 2014, 19, 14139-14194. (58) Britton, J.; Raston, C. L.; Weiss, G. A. Rapid Protein Immobilization for Thin Film Continuous Flow Biocatalysis. Chem. Comm. 2016, 52, 10159-10162. (59) Batalla, P.; Fuentes, M.; Mateo, C.; Grazu, V.; Fernandez-Lafuente, R.; Guisan, J. M. Covalent Immobilization of Antibodies on Finally Inert Support Surfaces Through Their Surface Regions Having the Highest Densities in Carboxyl Groups. Biomacromolecules 2008, 9, 22302236, DOI: 10.1021/bm8003594. (60) Poverenov, E.; Li, M.; Bitler, A.; Bendikov, M. Major Effect of Electropolymerization Solvent on Morphology and Electrochromic Properties of PEDOT Films. Chem. Mater. 2010, 22, 4019-4025. (61) Heim, M.; Reculusa, S.; Ravaine, S.; Kuhn, A. Engineering of Complex Macroporous Materials Through Controlled Electrodeposition in Colloidal Superstructures. Adv. Funct. Mater. 2012, 22, 538–545. (62) Cheng, Z.; Wang, E.; Yang, X. Capacitive Detection of Glucose Using Molecularly Imprinted Polymers. Biosens. Bioelectron. 2001, 16, 179-185.


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(63) Tamboli, V. K.; Bhalla, N.; Jolly, P.; Bowen, C. R.; Taylor, J. T.; Bowen, J. L.; Allender, C. J.; Estrela, P. Hybrid Synthetic Receptors on MOSFET Devices for Detection of Prostate Specific Antigen in Human Plasma. Anal. Chem. 2016, 88, 11486-11490. (64) Roushani, M.; Valipour, A. Using Electrochemical Oxidation of Rutin in Modeling a Novel and Sensitive Immunosensor Based on Pt Nanoparticle and Graphene–Ionic liquid–Chitosan Nanocomposite to Detect Human Chorionic Gonadotropin. Sens. Actuators B 2016, 222, 11031111. (65) Mao, L.; Yuan, R.; Chaia, Y.; Zhuo, Y.; Yang, X. A New Electrochemiluminescence Immunosensor based on Ru(bpy)32+-doped TiO2 Nanoparticles Labeling for Ultrasensitive Detection of Human Chorionic Gonadotrophin. Sens. Actuators B 2010, 149, 226-232. (66) Zhang, B.; Mao, Q.; Zhang, X.; Jiang, T.; Chen, M.; Yu, F.; Fu, W. A Novel Piezoelectric Quartz Micro-array Immunosensor Based on Self-assembled Monolayer for Determination of Human Chorionic Gonadotropin. Biosens. Bioelectron. 2004, 19, 711-720. (67) Xia, N.; Wang, X.; Liu, L. A Graphene Oxide-Based Fluorescent Method for the Detection of Human Chorionic Gonadotropin. Sensors 2016, 16, 1699. (68) Teixeira, S.; Conlan, R. S.; Guya, O. J.; Sales, M. G. F. Label-free Human Chorionic Gonadotropin Detection at Picogram Levels Using Oriented Antibodies Bound to Graphene Screen-Printed Electrodes. J. Mater. Chem. B 2014, 2, 1852-1865.


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Scheme 1. (A) cartoon and (B) surface 3D view of hCG and (C) amino acids sequence of α and β subunits providing information of secondary protein folding and posttranslational sugar modification of the protein (http://www.rcsb.org/structure/1HRP).30 Amino acids labeled with red (K – lysine) are able to bind functional monomer bearing carboxylic acid. Amino acids labeled with green (D – aspartic acid, E – glutamic acid) are able to bind functional monomer bearing amine.


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Scheme 2. Steps of semi-covalent and surface imprinting of hCG in a poly(2.3’-bithiophene) inverse opal film.


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Figure 1. Compression isotherm of 500 nm diameter silica nanobeads, surface functionalized with (3-aminopropyl)triethoxysilane. Barrier speed was 10 cm2/min.


31


A

B 1

2

0.92

1.04

0.90

Current , mA

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1.02

0.88 1.00

0.86 0.98 0 10 20 30 40

Time, s

0

20 40 60

Time, s

Figure 2. (A) Changes of current during potentiostatic deposition of 2,3’-(bithiophene) on Auglass slides covered with colloidal crystals (1) containing hCG protein derivatized with functional monomers and (2) unmodified. The propylene carbonate solution containing 0.5 M 2,3’bithiophene and 3.5 M LiClO4 was used for this deposition. (B) SEM images of cross-section view (1, 3, 5) and top view (2, 4, 6) of (1, 2) colloidal crystal composed of four layers of 500 nm silica nanobeads, (3, 4) macroporous MIP film, and (5, 6) macroporous NIP film. During deposition of all films the charge passed through the working electrode was Q = +70 mC.


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A

B I

1600

1000

N 1sB

500

N 1sA

II

400

N 1sA

N 1sB

1000 500 1500

III

1000

N 1sA

N 1sB

500 1500

IV

N 1sA

1000

S 2p1A S 2p1B

0 1600

S 2p3B S 2p3A

IV

S 2p1A

1200 800 400 0 1600

V

1200

500 1500

S 2p3A

800

-1

-1

1500

III

1200

Intensity , counts s

1500

Intensity , counts s

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


V

1000

402

S 2p1A

400

500 404

S 2p3A

800

N 1sA

N 1sB

400

398

396

0

170

Binding energy, eV

169

168

167

166

165

164

163

162

Binding energy, eV

Figure 3. (A) The XPS spectra recorded in the N 1s electron binding energy region for (I) the colloidal crystal functionalized with 3-aminopropyltriethoxysilane, (II) the colloidal crystal with immobilized hCG, (III) the colloidal crystal modified with functional monomer derivatized hCG, (IV) the hCG templated MIP film deposited by electropolymerization on an Au-coated glass slide previously modified with a colloidal crystal of SiO2 beads with the functional monomer derivatized hCG, before and (V) after colloidal crystal removal and extraction of the hCG template. (B) The XPS spectra recorded in the S 2p electron binding energy region for (III) the colloidal crystal modified with functional monomer derivatized hCG, (IV) the hCG templated MIP film deposited by electropolymerization on an Au-coated glass slide previously modified with a colloidal crystal of SiO2 beads with the functional monomer derivatized hCG, before and (V) after colloidal crystal removal and extraction of the hCG template. Assignments of nitrogen atom electrons and sulphur atom electrons for deconvoluted peaks are indicated in the spectra.


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Figure 4. (A) The capacitance changes of macroporous MIP with time after injection of hCG of different concentrations. (B) the capacitance changes of macroporous NIP with time after injection of hCG of different concentrations. (C) The capacitance changes of macroporous MIP with time after injection of equimolar amounts of LH and FSH of different concentrations. (D) Corresponding calibration curves of capacitive chemosensors with macroporous MIP film (1, 2, 3, 4) and macroporous NIP film (5). Curve 1, 2, 3, and 4 shows calibration curve of hCG, HSA, cytochrome c, myoglobin, respectively. All binding experiments were performed in 10 mM carbonate buffer (pH = 10). The final concentration of hCG, FSH or LH is indicated at arrow. For capacitance determination the electrode was kept at a constant potential (0.2 V vs. Ag|AgCl pseudo-reference electrode) and frequency (10 Hz).


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Figure 5. (A) Calibration plots for the EG-FET chemosensor with the extended gate region modified with macroporous, hCG templated MIP with respect to (1) hCG, (2) myoglobin (3) cytochrome c and (4) HSA. (B) Calibration plots for the EG-FET chemosensors with respect to the target hCG analyte with (1) macroporous, hCG templated MIP film and (2) macroporous NIP film. (C) Interference study performed in the presence of common biomolecules with macroporous MIP modified EG-FET chemosensors. All binding experiments were performed in 10 mM carbonate buffer (pH = 10). (D) Calibration plots for the EG-FET chemosensor with macroporous hCG templated MIP with respect to hCG hormone recorded in (1) 10 mM carbonate buffer and (2) in control Nortrol serum sample diluted with 10 mM carbonate buffer. The Pt reference electrode was polarized to the gate voltage, VR= 2.0 V.


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ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

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Table 1. Comparison of analytical parameters of bio- and chemosensor reported for hCG hormone protein Description of recognition material

Method of transduction

Limit of detection

Linear dynamic concentration range

Ref.

Antibody specific to hCG attached to polyaniline modified graphene-(screen printed electrode)

Impedimetry

~7.24 fM 0.286 pg/mL ~0.003 mIU/mL

~25.3 fM – 1.27 nM 0.001 – 50 ng/mL ~0.01 – 538 mIU/mL

68

Antibody specific to hCG immobilized over PtNPs, chitosan, graphene, and ionic liquid composite modified glassy carbon electrode

Differential pulse voltammetry

~0.82 fM ~0.032 pg/mL 0.00035 mIU/mL

~2.49 fM – 5.0 pM ~0.098 – 196.78 pg/mL 0.00106 – 350 mIU/mL

64

Antibody specific to hCG attached to AuNPs and Ru-Nafion-TiO2 NP assembled in a sandwich-type assays

Electrochemiluminescence

~16.45 fM ~0.65 pg/mL 0.007 mIU/mL

~47 fM – 58.75 pM ~1.86 pg/mL – 2.32 ng/mL 0.02 – 25 mIU/mL

65

Antibody specific to hCG immobilized on the quartz crystal resonator

Piezoelectic microgravimetry

-

~5.88 pM – 1.18 nM ~232 pg/mL – 46.41 ng/mL 2.5 – 500 mIU/mL

66

Peptide aptamer (PPLRINRHILTR) specific to hCG attached to AgNPs

Linear sweep voltammetry

~940 fM ~37.13 pg/mL 0.4 mIU/mL

~2.35 - 470 pM ~92.8 pg/mL – 18.56 ng/mL 1 – 200 mIU/mL

46

Peptide aptamer (PPLRINRHILTR) specific to hCG attached to graphene oxide

Surface plasmon resonance

0.065 nM ~2.57 ng/mL ~27.66 mIU/mL

0.065 – 8.32 nM ~2.57 – 328.64 ng/mL ~27.66 – 3540 mIU/mL

45

Peptide aptamer (PPLRINRHILTR) specific to hCG attached to graphene oxide

Fluorescence

~47 pM ~1.86 ng/mL 20 mIU/mL

~117.5 pM – 47 nM ~4.64 ng/mL – 1.86 μg/mL 50 mIU/mL – 20 IU/mL

67

Peptide aptamer (PPLRINRHILTR) specific to hCG attached to AuNPs

Spectrophotometry (Colorimetry)

~35.25 pM 1.5 ng/mL 15 mIU/mL

~35.25 pM – 2.35 nM ~1.5 – 92.83 ng/mL 15 – 1000 mIU/mL

47


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Surface imprinted MIP film deposited on chitosan and MWCNTs modified glassy carbon electrode

Differential pulse voltammetry

~0.89 fM 0.035 pg/mL ~0.00038 mIU/mL

~12.66 fM – 6.33 nM 0.0005 – 250 ng/mL ~0.005 – 2693.24 mIU/mL

48

Macroporous semi-covalently surface imprinted MIP

Extended gate field-effect transistors Capacitive impedimetry

0.8 - 50.6 fM ~0.032 – 1.99 pg/mL 0.00034 – 0.02153 mIU/mL 0.17 – 2.30 fM ~0.007 – 0.091 pg/mL 7.2×10-5 – 0.00097872 mIU/mL

This work

Macroporous semi-covalently surface imprinted MIP

0.8 fM ~0.032 pg/mL 0.00034 mIU/mL 0.17 fM ~0.007 pg/mL 7.2×10-5 mIU/mL

This work


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Facile Fabrication of Surface Imprinted Macroporous Film for

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